Abstract: Pilot Project In humans, 10-20% of infertile men are azoospermic, meaning that they lack spermatozoa. Of the two types, non-obstructive (no blockage) azoospermia, in many cases, is idiopathic. Production of mature sperm involves complex developmental processes, ultimately generating specialized cells for conception. Amongst these processes includes epigenetic programming. While prenatal prospermatogonial genome-scale DNA methylation programming has been well studied, the epigenetic landscape of postnatal testicular cells has not. Current evidence suggests that aberrant epigenetic modifications may be one mechanism that leads to idiopathic, non-obstructive azoospermia. However, this may relate to cell composition rather than etiologies of spermatogenic failure. To distinguish between these possibilities, studies are required to determine whether the epigenetic landscape of specific spermatogonial and Sertoli cells is altered in non-obstructive azoospermia. In Aim 1, we hypothesize that aberrant epigenetic landscapes in postnatal spermatogonia play a role in azoospermia due to etiology rather than whole testes cell composition. To test this hypothesis, we will use two genetic mouse models with azoospermia to delineate whether DNA methylation and chromatin accessibility are compromised. The first model will eliminate all de novo methyltransferase activity using conditional Dnmt3a/Dnmt3b double mutants targeting the germ cell compartment, while the second model will employ the androgen receptor knockout (SCARKOtm2.1) targeting the Sertoli compartment. In Aim 2, we hypothesis that men with non-obstructive azoospermia will harbor aberrant DNA methylation and chromatin accessibility within either the germ cell or Sertoli compartments. To test these hypothesis, we will produce DNA methylation, chromatin accessibility and expression reference sequences from normal undifferentiated and differentiating spermatogonia as well as Sertoli cells, following which we will compare the profile of these cells to those from mutant mice, or men with maturation arrest or Sertoli cell only syndrome. For both aims, cells will be isolated using the same enrichment strategy, and DNA methylation, chromatin accessibility and expression landscapes for undifferentiated spermatogonia, differentiating spermatogonia and Sertoli cells will be generated using NMT-seq (nucleosome, methylation and transcription sequencing), allowing comparative analysis between mouse and human. Overall, we will produce foundational data about the epigenetic landscape in germ cells and somatic cells of the testes and determine whether this landscape is altered in infertile mice and men.